A specific in vitro bioassay for measuring erythropoietin levels in human
serum and plasma
AW Wognum, V Lam, R Goudsmit and G Krystal
Terry Fox Laboratory, Cancer Control Agency of British Columbia, Vancouver,
Canada.
The accurate measurement of biologically active erythropoietin (Ep) in
human serum and plasma using present in vivo and in vitro bioassays is
difficult because of the presence of both inhibitors and non-Ep stimulators
of erythropoiesis. We have developed a simple procedure to quantitatively
purify Ep from serum and plasma for subsequent testing in the
phenylhydrazine-treated mouse spleen cell assay. The method involves
absorption of Ep to an immobilized high-affinity anti-Ep monoclonal
antibody and acid elution of the antibody-bound material. After
neutralization, the eluted EP is then tested directly in the in vitro
bioassay without interference by other serum proteins. By using magnetic
beads as a solid support for the antibody, washing and elution steps can be
performed rapidly and efficiently. Recoveries of Ep after this procedure
show very little sample-to-sample variation and are consistently between
45% and 55%, which is close to the maximum binding expected for the anti-Ep
antibody. Coupled with the 7.4-fold concentration that this procedure
affords, there is an overall increase in sensitivity of three- to fourfold,
which makes this assay suitable for accurately measuring Ep levels in
patients with below-average titers. Results with this magnetic bead assay
indicate that accurate and reproducible estimates for Ep levels in the
serum and plasma from healthy donors as well as from patients with
hematologic disorders can be obtained. Titers of biologically active Ep in
the sera from a group of patients with either leukemia or lymphoma were
found to be elevated, and the values correlated well with titers of
immunoreactive Ep measured in the Ep radioimmunoassay. Because of its
specificity and high sensitivity, the magnetic bead assay is a valuable
alternative to immunoassays for the measurement of elevated, normal, and
even subnormal Ep levels in human serum and plasma.
Volume 76,
Issue 7,
pp. 1323-1329,
10/01/1990
Copyright © 1990 by The American Society of Hematology
